Using temperature-sensitive murine sarcoma virus transformed rat fibroblasts and a mos-resistant revertant cell line (F-1), we have examined the ability of the serine/threonine kinase v-mos oncogene to activate the upstream activators of mitogen-associated protein kinase in transformed cells. Like MKK-1, MKK-2 is constitutively activated in mos-transformed cells. In cells which express v-mos in a temperature-dependent manner both MKK-1 and MKK-2 are activated within 1 hour of a shift to the permissive temperature. c-raf-1 is also constitutively activated in mos- transformed cells, but is not activated until 4 hours after the shift, suggesting its activation may be a secondary effect of mos. Constitutively-active MKK-1 and MKK-2 both transform normal rat fibroblasts and the F-1 revertant, consistent with the hypothesis that mos activation of MKK may be both necessary and sufficient for mos-induced transformation of rat fibroblasts. Murine retroviruses show a very low frequency of incorrect polyadenylation leading to read-through transcripts. We generated drug-selectable murine viruses containing mutations in the ..AATAAA.. polyadenylation signal and showed that the majority of cells infected by these viruses expressed read-through transcripts containing up to 1.2 kb of cell-derived sequences. Sequence analysis reveals that read-through transcripts consist of both unique and repetitive host-derived sequences. The growth and attachment-promoting activity present in media conditioned by SFDH, an NIH3T3 cell line which grows in serum-free media, is pelletable (150,000 g, 24 hours), associated with vesicle-like structures, and can be fractionated in 6M urea on a Superose-12 column.